Protein folding requires the existence of pathways to the native state. Although the contribution of amino acid residues to the thermodynamic stability of proteins has been intensively studied, very little is known about how amino acid sequences specify folding pathways. The proposed research is a combined molecular biological and biophysical approach to this problem. Because the complexity of the folding problem increases with chain length, the research will focus on one of the shortest sequences known to fold into a unique, stable structure without disulfide bonds: the 56 residue IgG binding domain of Peptostreptococcal Protein L. Extremely heavy mutagenesis protein L followed by selection for IgG binding using the phage display technology will be used to generate a database of very divergent sequences which adopt the same fold. Analysis of features conserved in the database should identify residues and interactions important in specifying the folding pathway. Determination of the folding times of a divergent subset of the sequences will provide insight into how sequence controls the selection and rate of traversal of kinetic pathways. The folding pathways of the most slowly folding mutants will be mapped using NMR methods. The sequence, rate and structure database together with the biophysical data on the folding pathway will be used to guide and constrain the development of a quantitative theory for the folding of this small protein. A detailed understanding of how amino acid sequence specifies tertiary structure in this simplest possible case should contribute to the understanding of the folding of more complex proteins.